The Early Steps in the Photocycle of a Photosensor Protein Sensory Rhodopsin I from Salinibacter ruber

Light absorption by the photoreceptor microbial rhodopsin triggers trans–cis isomerization of the retinal chromophore surrounded by seven transmembrane α-helices. Sensory rhodopsin I (SRI) is a dual functional photosensory rhodopsin both for positive and negative phototaxis in microbes. By making use of the highly stable SRI protein from Salinibacter ruber (SrSRI), the early steps in the photocycle were studied by time-resolved spectroscopic techniques. All of the temporal behaviors of the S_n←S₁ absorption, ground-state bleaching, K intermediate absorption, and stimulated emission were observed in the femto- to picosecond time region by absorption spectroscopy. The primary process exhibited four dynamics similar to other microbial rhodopsins. The first dynamics (τ₁ ∼ 54 fs) corresponds to the population branching process from the Franck–Condon region to the reactive (S₁^r) and nonreactive (S₁^nr) S₁ states. The second dynamics (τ₂ = 0.64 ps) is the isomerization process of the S₁^r state to generate the ground-state 13-cis form, and the third dynamics (τ₃ = 1.8 ps) corresponds to the internal conversion of the S₁^nr state. The fourth component (τ₃′ = 2.5 ps) is assignable to the J-decay (K-formation). This reaction scheme was further supported by the results of fluorescence spectroscopy. To investigate the protein response(s), the spectral changes of the tryptophan bands were monitored by ultraviolet resonance Raman spectroscopy. The intensity change following the K formation in the chromophore structure (τ ∼ 17 ps) was significantly small in SrSRI as compared with other microbial rhodopsins. We also analyzed the effect(s) of Cl^– binding on the ultrafast dynamics of SrSRI. Compared with a chloride pump Halorhodopsin, Cl^– binding to SrSRI was less effective for the excited-state dynamics, whereas the binding altered the structural changes of tryptophan following the K-formation, which was the characteristic feature for SrSRI. On the basis of these results, a primary photoreaction scheme of SrSRI together with the role of chloride binding is proposed.